Higher spatial resolution is required in the field of observation or measurement of a micro structure of a sample. One of methods for enhancing lateral resolution of the sample is as below. In the method, in order to form an image of light having a high spatial frequency component in light diffracted from the sample, a certain type of modulation is performed to diffracted light or illumination light in the proximity of a sample surface or in a position conjugate with the sample surface in an illumination optical system. Then demodulation corresponding to the performed modulation is performed in a position substantially conjugate with the sample surface in an image-formation optical system. A Lukosz method (W. Lukosz, “Optical systems with resolving powers exceeding the classical limit. II”, Journal of the Optical Society of America, Vol. 37, PP. 932 (1987)) (Non-Patent Document 1) is well known in days gone by as such a method, and recently there are methods disclosed in Japanese Patent Application Laid-Open No. 11-242189 (Patent Document 1) and U.S. Pat. RE38307 (Patent Document 2).
In the Lukosz method, diffraction gratings are disposed in the proximity of the sample and in a position substantially conjugate with the sample surface in the image-formation optical system, respectively, and the diffraction gratings are moved in a conjugate manner. The diffraction grating disposed in the proximity of the sample surface can cause the diffracted light, which cannot originally impinge on an objective lens of the image-formation optical system, to reach an image plane. The light diffracted by the diffraction grating disposed in the proximity of the sample surface is demodulated by the diffraction grating disposed in the position substantially conjugate with the sample surface in the image-formation optical system, and an image of the light is formed as an original diffraction component. The light having high spatial frequency component, which does not originally contribute to the image formation, reaches the image plane by means of the diffraction grating disposed in the proximity of the sample surface. As a result, spatial resolution higher than usual can be obtained.
A sixth embodiment disclosed in Japanese Patent Application Laid-Open No. 11-242189 is an example in which the Lukosz method is applied to a fluorescent observation device. In the optical system of the sixth embodiment disclosed in Japanese Patent Application Laid-Open No. 11-242189, illumination light emitted from a coherent light source is split by light beam splitting means such as a diffraction grating, and each of the illumination light beams is collected to a pupil position of an objective lens. Then, the illumination light beams are delivered from the objective lens as parallel light beams having different angles, and the parallel light beams overlap in the proximity of a sample, to form an interference fringe.
In the sixth embodiment disclosed in Japanese Patent Application Laid-Open No. 11-242189, spatial modulation is performed by the illumination light instead of disposing the diffraction grating in the proximity of the sample in the Lukosz method. Like the Lukosz method, advantageously the diffracted light including a spatial frequency component of shape information of the sample that cannot originally be transmitted only by the image-formation optical system can be involved in the image formation. Phases of the split illumination light beams are relatively modulated, and the interference fringe is moved on the sample to obtain plural images. Then an image can be formed by image arithmetic processing of the obtained images. Specifically the diffraction grating is moved perpendicular to an optical axis to perform the phase modulation, or a wedge-shape prism is inserted in one of optical paths of the illumination light and the wedge-shape prism is moved perpendicular to the optical axis to perform the phase modulation.
In the method disclosed in U.S. Pat. RE38307, after illumination light is introduced from a coherent light source using an optical fiber, the illumination light is split by light beam splitting means such as a diffraction grating, illumination light beams are collected to the pupil position of the objective lens, and an interference fringe is formed in the proximity of the sample. A high-frequency component of shape information of the sample that cannot originally be transmitted only by the image-formation optical system can be involved in the image formation by the illumination light modulated into a fringe shape. Similarly, plural images are obtained and subjected to image arithmetic processing to form an image of the sample. In the method disclosed in U.S. Pat. RE38307, when plural images are obtained in order to form an image of the sample, not only phase modulation is performed to the illumination light beams, but also an orientation of the interference fringe of the illumination light is changed. This is attributed to the following reason. The high-frequency component can be involved in the image formation only in a structure having the same direction as that of the interference fringe of the illumination light. Accordingly, in order to reproduce a shape of the two-dimensionally spread sample it is necessary to obtain plural images while changing the direction of the interference fringe and combine the plural images.
Patent Document 1: Japanese Patent Application Laid-Open No. 11-242189
Patent Document 2: U.S. Pat. RE38307
Non-Patent Document 1: Journal of the Optical Society of America, Vol, 37, PP. 932 (1967)
Non-Patent Document 2: M. G. L. Gustafsson, D. A. Agard, J. W. Sedat “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination”, Proceedings of the SPIE—The International Society for Optical Engineering (2000) vol. 3919, p. 141-50.
However, in the configuration of the conventional methods, because the phase modulation is performed by moving an element (such as a diffraction grating) that performs the phase modulation in a direction perpendicular to the optical axis, images cannot be obtained rapidly. Particularly, when a sample is a living biological sample, it is essential to obtain images rapidly. However, in the conventional methods, images cannot be obtained rapidly enough.
In the Lukosz method, it is necessary that the two diffraction gratings on the sample side and the imaging plane side be continuously moved while being synchronous with each other, which results in an extremely complicated mechanism.
In the method disclosed in U.S. Pat. RE38307, the diffraction grating is rotated in order to obtain two-dimensional super resolution. The diffraction grating is moved as follows on the assumption that the orientation of the interference fringe is changed and set to three directions an angle between adjacent two of which is 120°. That is, an image is obtained in a reference position, two images are obtained after having linearly moved the diffraction grating in the direction perpendicular to the optical axis using a linear actuator, the diffraction grating is returned to the optical axis position, an image is obtained after having rotated the diffraction grating by 120° using a rotary motor, two images are obtained after having moved the diffraction grating in the direction perpendicular to the optical axis, the diffraction grating is returned to the optical axis position, an image is obtained after having rotated the diffraction grating from a reference to 240° using the rotary motor, two images are obtained after having moved the diffraction grating in the direction perpendicular to the optical axis, the diffraction grating is returned to the optical axis position, and the diffraction grating is returned to the reference position using the rotary motor.
A total of nine images are obtained by a combination of the rotations and the shifts, and one sample image is obtained by the subsequent arithmetic processing. At this point, there are two cases, that is, the case in which the linear actuator including the diffraction grating is rotated and the case in which the diffraction grating alone is rotated while the linearly stepping direction of the diffraction grating is always set to one direction of the reference angle. In the former, because a mass of the object to be rotated is large, a large driving torque is required, and it is difficult to quickly stop the object. In the latter, it is necessary that the rotating angle be correctly measured to obtain the correct amount for stepping the diffraction grating. Therefore, a measurement and arithmetic time is required.
In the method disclosed in Japanese Patent Application Laid-Open No. 11-242189 in which the wedge-shape prism is inserted in one of the optical paths and moved in the direction perpendicular to the optical axis, it is necessary that the wedge-shape prism equipped with a moving mechanism be rotated according to the rotation of the two light beams, or it is necessary that the angle be measured to obtain the movement amount. Therefore, a stopping time or a measurement and arithmetic time is required.
In view of the foregoing, an object of the invention is to provide a super-resolution microscope device, in which spatially modulated illumination light is used to involve high-frequency components of the sample shape in the image formation and the spatial modulation is demodulated by arithmetic processing of obtained images to obtain a high-resolution sample image, and which can obtain the images rapidly.